Acquisition And Identification Of The FLAs Transgenic Plants In Rice

Arabinogalactan protein riched in Hyp(hydroxyproline)is a kind of protein polysaccharide which widely distribute in higher plants.All the sort of molecular are modified by a number of carbohydrate side chains containing lots of arabinose and galactan.It has proved that AGPs play important roles in plant growth and development,such as cell division and cell differentiation,pattern formation of tissue and organ,signal transduction among cells,responsing to hormone,stress resistance and et al.We used callus formed by seed embryos of rice variety Japonica as original material.14 CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)gene edit vector with the realated to OsFLA genes and 1 RNAi vector with the related to OsAGP gene were constructed succesfully and transformed into callus by using the technology of Agrobacterium tumefaciens-mediated transformation.By means of genomic level identification and sequencing technology,we had identified some positive transgenic plants,which layed the foundation of intensive study to OsFLA and OsAGP.In the research,the results obtained are as follows:1.A large number of callus tissue with good quality were induced by using seed embryos of wild type rice Japonica var.Nipponbare as materials;With repeat experiments,various parameters affecting callus tissues and transforming efficiency of the transgenic methodology system in our laboratory were optimized.(1)In the same conditions,when seeds of Nipponbare were induced into callus tissue,the mature seeds were easier to be contaminated than the inmature seeds.(2)When seeds of Nipponbare were induced into callus tissue by using sterile water cleaned for 6 times;75%ethyl alcohol cleaned for 3 minutes;0.1%HgCl handled for 25 minutes,less pollution rate will occur(3)When the additive amount of 2,4-D was 2.0mg/L or 2.4mg/L,frequency of occurrence of the callus tissue could reach the highest value.(4)When the additive amount of L-proline was 0.6g/L or 1.0g/L,frequency of occurrence of the callus tissue could reach the highest value.(5)When agrobacterium-mediated transforming and co-culture were conducted,frequency of occurrence of the resistant callus tissue could reach the highest in the callus tissues which had been subcultured for 12 days and the transformed callus tissues with the bacterium co-cultured for 3 days.(6)When the cleaning callus tissue were conducted by using carbenicillin with 0.75g/L or 1.00g/L,the best inhibitory effect could be obtained.2.In the research,15 vectors constructed successfully by our lab were transformed.Among them,14 were CRISPR/Cas9 mediated OsFLA gene knock-out vectors;while the other was OsAGP12 RNAi vector.In total,we acquired positive callus tissues of 10 vectors.Through genomic identification,we acquired transgenic lines in T0 generation of 7 with CR-OsFLA9,1 with CR-OsFLA19(1),20 with CR-OsFLA19,16 with CR-OsFLA20,7 with CR-OsFLA21,12 with CR-OsFLA22,9 with CR-OsFLA25,4 with CR-OsELA2,5 with CR-OsELA3 and 1 with Si-OsAGP12.3.DNA mutation target site's sequencing identification was conducted and 12 transgenic lines were obtained,in which there were 7 different mutational modes.